Transkriptionsregulation der Pyruvatdehydrogenase-Kinase 4 (PDK4), einem zentralen Schalter des Glukosestoffwechsels, durch Zelladhäsion und onkogene Signalwege beim humanen Ovarialkarzinom

Das Ovarialkarzinom ist die gynäkologische Krebserkrankung mit der höchsten Letalität, was vor allem durch die passive Metastasierung über den Peritonealraum bedingt ist. Dabei breiten sich Tumorzellen als Einzelzellen oder mehrzellige Sphäroide über die Peritonealflüssigkeit aus, um an anderer Stelle Metastasen auszubilden. Der damit einhergehende Adhärenzverlust dieser Zellen ist demnach von großer klinischer Relevanz und bedarf weiterer funktioneller Analysen. Im Rahmen dieser Arbeit konnte mit PDK4 erstmals ein Gen in primären humanen Ovarialkarzinomzellen und in der Modellzelllinie SKOV-3 identifiziert werden, dessen Expression stark von Adhärenz und Adhärenzverlust beeinflusst wird. Die mit dem Adhärenzverlust einhergehende Induktion scheint mit dem glykolytischen Metabolismus dieser Zellen funktionell in Verbindung zu stehen, im Einklang mit der zentralen Rolle von PDKs als negative Regulatoren der Pyruvatdehydrogenase und somit der Energiegewinnung aus Glukose durch oxidative Phosphorylierung. Eine detaillierte Untersuchung der adhärenzvermittelten PDK4-Regulation zeigte, dass Interaktionen zwischen Komponenten der Extrazellulärmatrix und Integrinen dafür verantwortlich sind. Der weitere Fokus der vorliegenden Arbeit lag auf der detaillierten mechanistischen Aufklärung dieser transkriptionellen Kontrolle und die damit verbundene Identifizierung der verantwortlichen Signalwege anhand der Modellzelllinie SKOV-3. Hierzu wurde nach pharmakologischer Modulation bestimmter Signalwege oder einer RNAi-vermittelten Inhibition spezifischer Signalproteine die Phosphorylierung kritischer Signalkomponenten, Chromatinbindung und Regulation von PDK4 untersucht. Die hierbei erzielten Ergebnisse deuten auf eine Kooperation mehrerer Signalwege bei der Regulation des PDK4-Gens durch Adhärenzverlust hin. Hierzu zählen der SRC-PI3K-AKT-Signalweg, die GSK3-β-Catenin-Signalkaskade, der Estrogenrezeptor, der Transkriptionsfaktor C/EBPβ und Umstrukturierungen des Aktinzytoskeletts. Über die adhärenzvermittelte PDK4-Regulation hinaus liefern die Ergebnisse dieser Arbeit ein Bild zur Transkriptionsregulation des PDK4-Gens durch tumorbiologisch relevante Signale im humanen Ovarialkarzinom. So führen die Aktivierung des Adenylylcyclase-cAMP-PKA-Signalwegs, die Inhibition des SRC-PI3K-AKT-Signalwegs sowie Estrogen, Glukokortikoid und Zell-Zell-Kontakte zu einer Induktion der PDK4-Expression. Die komplexe Regulation von PDK4, insbesondere auch durch onkogene Signalwege, und die zentrale Funktion im Glukosemetabolismus unterstreichen die essentielle Rolle von PDK4 im Tumormetabolismus und weisen auf PDK4 als mögliches therapeutisches Ziel bei der Behandlung des Ovarialkarzinoms hin

Negative plant–soil feedback predicts tree-species relative abundance in a tropical forest

The accumulation of species-specific enemies around adults is hypothesized to maintain plant diversity by limiting the recruitment of conspecific seedlings relative to heterospecific seedlings1,2,3,4,5,6. Although previous studies in forested ecosystems have documented patterns consistent with the process of negative feedback7,8,9,10,11,12,13,14,15,16, these studies are unable to address which classes of enemies (for example, pathogens, invertebrates, mammals) exhibit species-specific effects strong enough to generate negative feedback17, and whether negative feedback at the level of the individual tree is sufficient to influence community-wide forest composition. Here we use fully reciprocal shade-house and field experiments to test whether the performance of conspecific tree seedlings (relative to heterospecific seedlings) is reduced when grown in the presence of enemies associated with adult trees. Both experiments provide strong evidence for negative plant–soil feedback mediated by soil biota. In contrast, above-ground enemies (mammals, foliar herbivores and foliar pathogens) contributed little to negative feedback observed in the field. In both experiments, we found that tree species that showed stronger negative feedback were less common as adults in the forest community, indicating that susceptibility to soil biota may determine species relative abundance in these tropical forests. Finally, our simulation models confirm that the strength of local negative feedback that we measured is sufficient to produce the observed community-wide patterns in tree-species relative abundance. Our findings indicate that plant–soil feedback is an important mechanism that can maintain species diversity and explain patterns of tree-species relative abundance in tropical forests

Transkriptionsregulation der Pyruvatdehydrogenase-Kinase 4 (PDK4), einem zentralen Schalter des Glukosestoffwechsels, durch Zelladhäsion und onkogene Signalwege beim humanen Ovarialkarzinom

Das Ovarialkarzinom ist die gynäkologische Krebserkrankung mit der höchsten Letalität, was vor allem durch die passive Metastasierung über den Peritonealraum bedingt ist. Dabei breiten sich Tumorzellen als Einzelzellen oder mehrzellige Sphäroide über die Peritonealflüssigkeit aus, um an anderer Stelle Metastasen auszubilden. Der damit einhergehende Adhärenzverlust dieser Zellen ist demnach von großer klinischer Relevanz und bedarf weiterer funktioneller Analysen. Im Rahmen dieser Arbeit konnte mit PDK4 erstmals ein Gen in primären humanen Ovarialkarzinomzellen und in der Modellzelllinie SKOV-3 identifiziert werden, dessen Expression stark von Adhärenz und Adhärenzverlust beeinflusst wird. Die mit dem Adhärenzverlust einhergehende Induktion scheint mit dem glykolytischen Metabolismus dieser Zellen funktionell in Verbindung zu stehen, im Einklang mit der zentralen Rolle von PDKs als negative Regulatoren der Pyruvatdehydrogenase und somit der Energiegewinnung aus Glukose durch oxidative Phosphorylierung. Eine detaillierte Untersuchung der adhärenzvermittelten PDK4-Regulation zeigte, dass Interaktionen zwischen Komponenten der Extrazellulärmatrix und Integrinen dafür verantwortlich sind. Der weitere Fokus der vorliegenden Arbeit lag auf der detaillierten mechanistischen Aufklärung dieser transkriptionellen Kontrolle und die damit verbundene Identifizierung der verantwortlichen Signalwege anhand der Modellzelllinie SKOV-3. Hierzu wurde nach pharmakologischer Modulation bestimmter Signalwege oder einer RNAi-vermittelten Inhibition spezifischer Signalproteine die Phosphorylierung kritischer Signalkomponenten, Chromatinbindung und Regulation von PDK4 untersucht. Die hierbei erzielten Ergebnisse deuten auf eine Kooperation mehrerer Signalwege bei der Regulation des PDK4-Gens durch Adhärenzverlust hin. Hierzu zählen der SRC-PI3K-AKT-Signalweg, die GSK3-β-Catenin-Signalkaskade, der Estrogenrezeptor, der Transkriptionsfaktor C/EBPβ und Umstrukturierungen des Aktinzytoskeletts. Über die adhärenzvermittelte PDK4-Regulation hinaus liefern die Ergebnisse dieser Arbeit ein Bild zur Transkriptionsregulation des PDK4-Gens durch tumorbiologisch relevante Signale im humanen Ovarialkarzinom. So führen die Aktivierung des Adenylylcyclase-cAMP-PKA-Signalwegs, die Inhibition des SRC-PI3K-AKT-Signalwegs sowie Estrogen, Glukokortikoid und Zell-Zell-Kontakte zu einer Induktion der PDK4-Expression. Die komplexe Regulation von PDK4, insbesondere auch durch onkogene Signalwege, und die zentrale Funktion im Glukosemetabolismus unterstreichen die essentielle Rolle von PDK4 im Tumormetabolismus und weisen auf PDK4 als mögliches therapeutisches Ziel bei der Behandlung des Ovarialkarzinoms hin

Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ

<div><p>The peroxisome proliferator-activated receptor subtypes PPARα, PPARβ/δ, PPARγ are members of the steroid hormone receptor superfamily with well-established functions in transcriptional regulation. Here, we describe an unexpected cytoplasmic function of PPARβ/δ. Silencing of PPARβ/δ expression interferes with the expression of a large subset of interleukin-1β (IL-1β)-induced target genes in HeLa cells, which is preceded by an inhibition of the IL-1β-induced phosphorylation of TAK1 and its downstream effectors, including the NFκBα inhibitor IκBα (NFKBIA) and the NFκBα subunit p65 (RELA). PPARβ/δ enhances the interaction between TAK1 and the small heat-shock protein HSP27, a known positive modulator of TAK1-mediated IL-1β signaling. Consistent with these findings, PPARβ/δ physically interacts with both the endogenous cytoplasmic TAK1/TAB1 complex and HSP27, and PPARβ/δ overexpression increases the TAK1-induced transcriptional activity of NFκB. These observations suggest that PPARβ/δ plays a role in the assembly of a cytoplasmic multi-protein complex containing TAK1, TAB1, HSP27 and PPARβ/δ, and thereby participates in the NFκB response to IL-1β.</p></div

Effect of PPARβ/δ depletion on global transcriptional response to IL-1β.

<p>(<b>A</b>) Diagrammatic representation of IL-1β target genes (threshold ≥2-fold; <i>n</i> = 113) showing a reduced induction by IL-1β (threshold ≥1.8-fold; <i>n</i> = 55) or no significant effect on induction (threshold ≤1.4-fold; <i>n</i> = 32) after PPARβ/δ depletion. HeLa cells were treated with control siRNA <i>(</i>si-con) or <i>PPARD</i>-directed siRNA (si-PPARD) followed by IL-1β (10 ng/ml) for 1 h (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s001" target="_blank">Figure S1</a> for knockdown efficiency). Expression patterns were determined by microarray analyses and genes showing a ≥2-fold regulation were identified (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s015" target="_blank">Datasets S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s016" target="_blank">S2</a>). The observed regulation was verified by RT-qPCR, as exemplified for the genes listed in the boxed areas and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s002" target="_blank">Figures S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s003" target="_blank">S3</a>. (<b>B</b>) Scatter plot showing the IL-1β response of individual genes with or without <i>PPARD</i> silencing (microarray data from panel A). The dashed line shows the ideal position of genes theoretically unaffected by si-PPARD. Blue data points: effect ≤1.4-fold; red data points: effect ≥1.8-fold. (<b>C</b>), (<b>D</b>) Effect of PPARβ/δ depletion on the time course of the IL-1β-mediated induction of the <i>IL6</i> (C) and <i>IL8</i> (D) gene determined by RT-qPCR. (<b>E</b>) Effect of PPARβ/δ depletion on IL-1β-induced IL-6 secretion in HeLa cells determined by ELISA (1 h and 4 h stimulation with IL-1β). Values represent averages ±SD (<i>n</i> = 3). ***, **, *significant difference between si-con and si-PPARD-treated cells (<i>p</i><0.001, <i>p</i><0.01, <i>p</i><0.05 by t-test).</p

Modulation of p65 binding to NFκB target genes <i>in vivo</i> by PPARβ/δ.

<p>HeLa cells were treated with IL-1β and siRNAs as indicated and ChIP assays were performed with antibodies against PPARβ/δ (green), RXR (white) or p65 (red) or control IgG (grey). PCR primers were designed to detect the NFκB binding sites of the <i>IL6</i> (<b>A</b>), <i>IL8</i> (<b>B</b>), <i>BCL3</i> (<b>C</b>) and <i>CXCL10</i> (<b>D</b>) genes, the triple-PPRE of the ANGPTL4 gene (<b>E</b>) or an irrelevant genomic control region (<b>F</b>). Relative amounts of amplified DNA in immunoprecipitates were calculated by comparison with 1% of input DNA. Results are expressed as % input and represent averages of triplicates (± S.D). ***, **, *significant differences between si-con and si-PPARD-treated cells (<i>p</i><0.001, <i>p</i><0.01, <i>p</i><0.05 by t-test).</p

Interaction of endogenous PPARβ/δ with HSP27, TAK1 and TAB1.

<p>Untransfected HEK293T cells were treated with formaldehyde to stabilize protein interactions following the protocol for ChIP analyses. Cell extracts were prepared and immuneprecipitations were carried out with either irrelevant IgG or with antibodies against PPARβ/δ. RXR, HSP27, TAK1 or TAB1 (IP). Immunoblotting was performed with PPARβ/δ-specific antibodies. Antibodies against the established PPAR heterodimerization partner RXR were included as a positive control. The PPARβ/δ-HSP27 co-immunoprecipitation was abolished after pretreatment of the cell with HSP27 siRNA, confirming its specificity (not shown). The two rightmost lanes represent untreated extracts from HCT116 cells with intact (+/+) or disrupted (−/−) <i>PPARD</i> alleles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011-Park2" target="_blank">[34]</a> to allow for unambiguous identification of the PPARβ/δ band. *, non-specific band.</p

Complex formation of PPARβ/δ with TAK1/TAB1.

<p>(<b>A</b>) Co-immunoprecipitation of PPARβ/δ with TAK1 and TAB1. HEK293IL-1R cells were transfected with expression vectors for MYC-tagged TAB1, GFP-tagged TAK1 and FLAG-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against TAB1, TAK1 or FLAG. Input lanes were loaded with 50 µg protein (3% of the amount used for IPs). (<b>B</b>) Co-immunoprecipitation of PPARβ/δ and TAK1. HEK293IL-1R cells were transfected with expression vectors for HA-tagged TAK1 and/or FLAG-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against FLAG, and immunoblotting with antibodies against TAK1 or FLAG. (<b>C</b>) Co-immunoprecipitation of PPARβ/δ and TRAF6. HEK293IL-1R cells were transfected with expression vectors for FLAG-tagged TRAF6 and/or CFP-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against GFP or TRAF6. (<b>D</b>) Co-immunoprecipitation of PPARβ/δ and p65. HEK293IL-1R cells were transfected with expression vectors for FLAG-tagged PPARβ/δ and/or HA-tagged p65. Pulldown (PD) was carried out with an antibody against FLAG, and immunoblotting with antibodies against FLAG or HA. (<b>E</b>) Cytoplasmic interactions of FLAG-PPARβ/δ with endogenous TAK1. HEK293T cells were transfected with expression vectors for FLAG-tagged PPARβ/δ. Cells were fractionated into cytoplasmic and nuclear fractions, pulldown (PD) was carried out with an antibody against FLAG, and immunoblotting with antibodies against TAK1, FLAG and actin. (<b>F</b>) Subcellular localization of endogenous PPARβ/δ in HEK293T cells. Cytoplasmic and nuclear fractions were isolated and analyzed by immunoblotting with an antibody against PPARβ/δ. Antibodies against lactate dehydrogenase (LDH) and acetyl-Histon H3 were included in panels E and F to control for the purity of the cytoplasmic and nuclear fractions.</p

Functional and physical interaction of PPARβ/δ with HSP27.

<p>(<b>A</b>) Venn Diagram showing the overlaps of genes induced by IL-1β (blue; threshold ≥2-fold; <i>n</i> = 34; <i>n</i> = 113), downregulated by si-HSP27 (yellow; threshold ≥1.5-fold; <i>n</i> = 469) or downregulated by si-PPARD (red; threshold ≥1.8-fold; <i>n</i> = 155). HeLa cells were treated with control siRNA <i>(</i>si-con) or gene-specific siRNAs followed by IL-1β (10 ng/ml) for 1 h (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s008" target="_blank">S8</a> for knockdown efficiency). Expression patterns were determined by microarray analyses and genes showing a ≥1.5-fold regulation were identified (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s018" target="_blank">Dataset S4</a>). The observed regulation was verified by RT-qPCR, as exemplified for the genes listed in the boxed areas and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s009" target="_blank">Figure S9</a>. (<b>B, C</b>) Effect of HSP27 or PPARβ/δ depletion on the IL-1β-mediated induction of the <i>IL6</i> (B) and <i>IL8</i> (C) genes in HeLa cells determined by RT-qPCR. Values represent averages ±SD (<i>n</i> = 3). ***, **, *significant difference between si-con and si-PPARD-treated cells (p<0.001, <i>p</i><0.01, <i>p</i><0.05 by t-test). (<b>D</b>) Cytoplasmic interaction of PPARβ/δ and HSP27 detected by co-immunoprecipitation. HEK293T cells were transfected with expression vectors for HA-tagged HSP27 and/or FLAG-tagged PPARβ/δ. Cells were fractionated into cytoplasmic and nuclear fractions, pulldown (PD) was carried out with an antibody against FLAG and immunoblotting with anti-HA antibodies. (<b>E</b>) Immunoprecipitation of GFP-tagged TAK1 in complexes with FLAG-tagged PPARβ/δ, HA-tagged HSP27. HEK293IL-1R cells were transfected with the indicated expression vectors. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against TAK1, FLAG or HA. (<b>F</b>) Co-expression of PPARβ/δ enhances the interaction of HSP27 and TAK1. HEK293IL-1R cells were transfected with expression vectors for GFP-tagged TAK1, MYC-tagged TAB1, HA-tagged HSP27 and FLAG-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against TAK1, TAB1, FLAG and HSP27. (<b>G</b>) Same experiment as in panel F, except that MYC-TAB1 was omitted. (<b>H</b>) Immunoblot analysis of the indicated proteins at different time points after IL-1β stimulation of si-con and si-HSP27 treated HeLa cells. Details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone-0063011-g005" target="_blank">Figure 5B</a>. The siRNA effect on HSP27 protein levels in this experiment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s008" target="_blank">Figure S8</a>. (<b>I</b>) Effects of siRNA-mediated depletion of PPARβ/δ or/and HSP27 on the IL-1β-induced transcription of <i>IL6</i> (6 h stimulation with IL-1β). Values represent averages ±SD (<i>n</i> = 3). ***, **, *significant differences (<i>p</i><0.001, <i>p</i><0.01, <i>p</i><0.05 by t-test).</p

Effect of IL-1β on PPARβ/δ target genes.

<p>(<b>A</b>) Set (<i>n</i> = 51) of PPARβ/δ target genes (defined as genes upregulated by siRNA-mediated PPARβ/δ depletion) and empty subset of these genes affected by IL-1β (<i>n</i> = 0). HeLa cells were treated and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone-0063011-g001" target="_blank">Figure 1</a> (threshold ≥1.8-fold regulation; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s017" target="_blank">Dataset S3</a>). Verified genes are listed in the boxed area and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063011#pone.0063011.s004" target="_blank">Figure S4</a>. (<b>B</b>) Effect of IL-1β on the PPARβ/δ response of the <i>ANGPTL4</i> gene. Values represent averages ±SD (<i>n</i> = 3). ***, **, *significant difference between si-con and si-PPARD-treated cells (<i>p</i><0.001, <i>p</i><0.01, <i>p</i><0.05 by t-test).</p